The TLR7 agonists imiquimod and CL097 activate the NLRP3 inflammasome in myeloid cells through a route that dispenses with K+^+ efflux, the canonical trigger for NLRP3. The source work established that these imidazoquinolines inhibit the quinone oxidoreductase NQO2 and mitochondrial respiratory Complex I, inducing reactive oxygen species (ROS), protein thiol oxidation, and NEK7-dependent inflammasome activation, but left three linked mechanistic questions open: the full inventory of ROS-producing (and otherwise NLRP3-relevant) targets of these compounds; which downstream biochemical consequences — ROS magnitude and subcellular localization, NAD+^+/NADH and ATP perturbation, endolysosomal effects — are individually required and which combination of them triggers activation (Complex I inhibition alone, e.g., by rotenone, is insufficient); and the molecular chain connecting ROS-dependent thiol oxidation to inflammasome assembly, originally framed as ROS-to-NEK7 engagement. As of 2026 this route remains unreconstituted: partial redox relays have been identified in adjacent routes (GSTO1-1-mediated NEK7 deglutathionylation; JNK1-mediated NEK7 phosphorylation downstream of K+^+ efflux; ROS-oxidized cofilin-1 releasing NLRP3), and the NEK7 premise itself has been revised (NEK7 accelerates but is in principle dispensable for activation), while new chemical probes selectively targeting the K+^+-efflux-independent route are being developed precisely because its mechanism is unresolved. The problem is therefore open: reconstruct, at molecular resolution and with necessity/sufficiency evidence, the signal-transduction route from imidazoquinoline binding to NLRP3 inflammasome assembly that operates without K+^+ efflux.

Background

The NLRP3 inflammasome is a cytosolic multi-protein complex in innate immune cells (macrophages, monocytes, neutrophils) in which the sensor protein NLRP3 recruits the adaptor ASC and the cysteine protease caspase-1; upon activation, caspase-1 matures the pro-inflammatory cytokines interleukin-1β\beta (IL-1β\beta) and IL-18 and initiates pyroptosis, a lytic inflammatory cell-death program. NLRP3 responds to an unusually diverse set of stimuli — bacterial toxins, extracellular ATP, particulate crystals, viral RNA — which led to the proposal (Muñoz-Planillo et al., Immunity 2013) that these converge on a single proximal signal: efflux of potassium ions (K+^+ efflux) from the cytosol, which lowers intracellular K+^+ below a permissive threshold. Imiquimod (R837) and CL097 are imidazoquinoline small molecules, best known as agonists of the endosomal innate receptor Toll-like receptor 7 (TLR7); imiquimod is a licensed topical drug for viral skin lesions and cutaneous cancers. Beyond TLR7, both compounds induce NLRP3 activation in myeloid cells, and this activity is not blocked by clamping extracellular K+^+ at high concentration — i.e., it does not require K+^+ efflux, breaking the proposed common-trigger rule. The source work (Groß et al., Immunity 2016) identified two molecular targets underlying this activity: NQO2 (NRH:quinone oxidoreductase 2, a cytosolic flavoprotein quinone reductase) and mitochondrial respiratory Complex I (NADH:ubiquinone oxidoreductase, the first complex of the electron-transport chain, whose inhibition can cause electron leakage and reactive oxygen species, ROS, i.e., partially reduced oxygen species such as superoxide). Drug binding to both targets produced a burst of ROS, global oxidation of cysteine thiol groups on cytoplasmic proteins, and NLRP3 activation that required NEK7 (NIMA-related kinase 7, a kinase identified contemporaneously by He et al., Nature 2016, as an essential NLRP3 component acting downstream of K+^+ efflux). Crucially, pharmacological inhibition of Complex I alone (rotenone) did not activate NLRP3 under the same conditions, so Complex I inhibition is insufficient by itself; the authors proposed that metabolic consequences of Complex I inhibition (changes in NAD+^+/NADH redox state and ATP) and lysosome-disrupting (endolysosomal) effects of imiquimod, which is a lysosomotropic amphiphilic compound, might also contribute. In the decade since, the field has identified several redox-sensitive steps that modulate NLRP3 signaling in other activation routes — including deglutathionylation (removal of the glutathione adduct from an oxidized cysteine) of NEK7 at Cys253 by the enzyme GSTO1-1 (Hughes et al., 2019), phosphorylation of NEK7 by the kinase JNK1 downstream of K+^+ efflux (Xu et al., 2025), and ROS-dependent oxidation of the actin-severing protein cofilin-1, which otherwise restrains NLRP3 (Park et al., 2026) — and has determined structures of the NLRP3 complex. Yet the same period also revised the original NEK7 framework: in 2026 the source's own group showed that NEK7 accelerates but is in principle dispensable for NLRP3 activation, supporting two possible scenarios — either NEK7 accelerates a unique activation pathway, or a fast NEK7-dependent pathway operates in parallel with a slower NEK7-independent one. Reviews as late as 2026 still describe the NLRP3 activation mechanism as incompletely understood, and new chemical probes are being generated specifically for the K+^+-efflux-independent route. What is still missing is a connected, causal account of that route: every molecular target engaged by imiquimod/CL097 at pathway-relevant concentrations, which downstream biochemical perturbations are individually required and which combination of them triggers activation, and the molecular relay that transduces ROS and thiol oxidation into inflammasome assembly when the canonical K+^+-efflux signal is absent.

Problem Statement

Reconstruct, at molecular resolution and with causal (necessity and sufficiency) evidence, the K+^+-efflux-independent route by which the imidazoquinolines imiquimod and CL097 activate the NLRP3 inflammasome in myeloid cells. This comprises three jointly required deliverables that constitute one signal-transduction route from drug binding to inflammasome assembly: (1) the complete inventory of molecular targets engaged by imiquimod and CL097 at pathway-relevant concentrations in myeloid cells — that is, the concentrations at which these compounds activate NLRP3 with extracellular K+^+ clamped high, which in the source were approximately 70 μ\muM (15–20 μ\mug ml−1^{-1}) and up to 100 μ\muM in individual measurements — including any ROS-producing targets beyond NQO2 and mitochondrial Complex I, with their relative contributions to the ROS burst and to activation; the concentration window used and the detection criterion (binding affinity/occupancy or functional threshold) relative to which the inventory is claimed to be complete must be fixed in advance and stated explicitly rather than derived from the resulting hit list, and genuine pathway-relevant targets must be separated from nonspecific binding and from TLR7 signaling; (2) which downstream consequences are individually required for NLRP3 activation without K+^+ efflux and which combination of them is sufficient to trigger it — ROS of sufficient magnitude to oxidize cytoplasmic protein thiols, resolved as in the source between mitochondrial and cytoplasmic compartments, the consequences of Complex I dysfunction including the reduced NAD+^+/NADH ratio and ATP depletion that the source flagged only tentatively, and endolysosomal perturbation, which the source showed is not sufficient on its own but may contribute — together with an explanation of the source's finding that Complex I inhibition alone (e.g., rotenone) induces insufficient ROS and protein oxidation and therefore fails to activate NLRP3; where the source's own data leave a contribution uncertain, an answer may report that contribution as required, contributory or dispensable together with the supporting evidence; and (3) the molecular relay that couples ROS-dependent protein thiol oxidation to inflammasome assembly in this route — which proteins are oxidized or otherwise modified, in what order, and how these events converge on NLRP3 activation, where the original ROS-to-NEK7 framing must now accommodate the finding that NEK7 accelerates but is dispensable for activation: either scenario proposed by Wöhrle et al. 2026 is admissible — NEK7 accelerating a unique activation pathway, or a fast NEK7-dependent pathway operating in parallel with a slower NEK7-independent one — and the reconstructed relay must state which one the evidence supports. Necessity and sufficiency are to be established for an explicitly specified cellular system (cell type, species, priming state, and activation readout), with any dependence of the conclusions on these conditions reported rather than averaged away. The route must be supported by causal evidence rather than correlation: sufficiency in particular must be demonstrated for a combination of downstream perturbations in K+^+-clamped myeloid cells, and cannot be met by applying imiquimod or CL097 themselves; the classes of experiment by which necessity and sufficiency are established are not prescribed.

References

  1. Groß CJ, Mishra R, Schneider KS, Médard G, Wettmarshausen J, Dittlein DC, Shi H, Gorka O, Koenig PA, Fromm S, Magnani G, Ćiković T, Hartjes L, Smollich J, Robertson AAB, Cooper MA, Schmidt-Supprian M, Schuster M, Schroder K, Broz P, Traidl-Hoffmann C, Beutler B, Kuster B, Ruland J, Schneider S, Perocchi F, Groß O. K+ Efflux-Independent NLRP3 Inflammasome Activation by Small Molecules Targeting Mitochondria. Immunity 2016;45(4):761-773. DOI: 10.1016/j.immuni.2016.08.010. https://doi.org/10.1016/j.immuni.2016.08.010
  2. Muñoz-Planillo R, Kuffa P, Martínez-Colón G, Smith BL, Rajendiran TM, Núñez G. K+ efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 2013;38(6):1142-1153. DOI: 10.1016/j.immuni.2013.05.016. https://doi.org/10.1016/j.immuni.2013.05.016
  3. He Y, Zeng MY, Yang D, Motro B, Núñez G. NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux. Nature 2016;530(7590):354-357. DOI: 10.1038/nature16959. https://doi.org/10.1038/nature16959
  4. Hughes MM, Hooftman A, Angiari S, Tummala P, Zaslona Z, Runtsch MC, McGettrick AF, Sutton CE, Diskin C, Rooke M, Takahashi S, Sundararaj S, Casarotto MG, Dahlström JE, Palsson-McDermott EM, Corr SC, Mills KHG, Preston RJS, Neamati N, Xie Y, Baell JB, Board PG, O'Neill LAJ. Glutathione Transferase Omega-1 Regulates NLRP3 Inflammasome Activation through NEK7 Deglutathionylation. Cell Reports 2019;29(1):151-161.e5. DOI: 10.1016/j.celrep.2019.08.072. https://doi.org/10.1016/j.celrep.2019.08.072
  5. Xu J, Zhang L, Duan Y, Sun F, Odeh N, He Y, Núñez G. NEK7 phosphorylation amplifies NLRP3 inflammasome activation downstream of potassium efflux and gasdermin D. Science Immunology 2025;10(103):eadl2993. DOI: 10.1126/sciimmunol.adl2993. https://doi.org/10.1126/sciimmunol.adl2993
  6. Wöhrle S, Ćikovic T, Dufossez C, Neuwirt E, Puma E, Kraatz F, Kostina A, Gorka O, Kreutz C, Groß CJ, Groß O. NEK7 Accelerates NLRP3 Inflammasome Activation. Journal of Molecular Biology 2026:169960. DOI: 10.1016/j.jmb.2026.169960. https://doi.org/10.1016/j.jmb.2026.169960
  7. Park YH, Batu ED, Matthiasardottir B, Kim Y, Akkaya-Ulum YZ, Kastner DL, Chae JJ. Cofilin-1 is a redox-sensitive guard of the NLRP3 inflammasome. Nature Immunology 2026;27(5):937-948. DOI: 10.1038/s41590-026-02477-8. https://doi.org/10.1038/s41590-026-02477-8
  8. Xue C, Zhang Z, Ding Z, Xie G, Liu R, Wang F, Yu J, Wang T, Xue F, Song H, Chen Q, Qin Y. Total Synthesis of Euphopia B and Discovery of a Selective Inhibitor of Potassium Efflux-Independent NLRP3 Inflammasome Activation. Journal of the American Chemical Society 2026;148(15):16359-16366. DOI: 10.1021/jacs.6c02838. https://doi.org/10.1021/jacs.6c02838
  9. Jin X, Liu D, Zhou X, Luo X, Huang Q, Huang Y. Entrectinib inhibits NLRP3 inflammasome and inflammatory diseases by directly targeting NEK7. Cell Reports Medicine 2023;4(12):101310. DOI: 10.1016/j.xcrm.2023.101310. https://doi.org/10.1016/j.xcrm.2023.101310
  10. Chang SH, Wu CY, Chuang KC, Huang SW, Li ZY, Wang ST, Lai ZL, Chang CC, Chen YJ, Wong TW, Kao JK, Shieh JJ. Imiquimod Accelerated Antitumor Response by Targeting Lysosome Adaptation in Skin Cancer Cells. Journal of Investigative Dermatology 2021;141(9):2219-2228.e8. DOI: 10.1016/j.jid.2021.01.034. https://doi.org/10.1016/j.jid.2021.01.034
  11. Shin HJ, Kim IS, Kim JK, Jo EK. Molecular mechanisms of NLRP3 inflammasome activation. Experimental & Molecular Medicine 2026;58(3):650-663. DOI: 10.1038/s12276-026-01656-9. https://doi.org/10.1038/s12276-026-01656-9